Anti-diabetic agents

ABSTRACT

The present invention provides compounds of formula (I)  
                 
 
     the prodrugs thereof, and the pharmaceutically acceptable salts of the compounds and prodrugs, wherein R′, R″, R′″, and Z are as defined herein; pharmaceutical compositions thereof; and uses thereof in treating diabetes, insulin resistance, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, cataracts, hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, and tissue ischemia.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/424,627 filed Nov. 7, 2003.

BACKGROUND OF THE INVENTION

[0002] The invention relates to certain substitutedN-(indole-2-carbonyl)amides and 6H-thieno[2,3-b]pyrrole-5-carboxamideswhich are antidiabetic agents and, as such, are useful in the treatmentof diabetes, insulin resistance, diabetic neuropathy, diabeticnephropathy, diabetic retinopathy, cataracts, hyperglycemia,hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia,atherosclerosis, and tissue ischemia, particularly myocardial ischemia.This invention also relates to methods of using such compounds in thetreatment of the above diseases in mammals, especially humans, and topharmaceutical compositions useful therefor.

[0003] In spite of the early discovery of insulin and its subsequentwidespread use in the treatment of diabetes, and the later discovery ofand use of sulfonylureas, biguanides and thiazolidenediones, such astroglitazone, rosiglitazone or pioglitazone, as oral hypoglycemicagents, the treatment of diabetes remains less than satisfactory.

[0004] The use of insulin requires multiple daily doses, usually byself-injection. Determination of the proper dosage of insulin requiresfrequent estimations of the sugar in urine or blood. The administrationof an excess dose of insulin causes hypoglycemia, with effects rangingfrom mild abnormalities in blood glucose to coma, or even death.Treatment of non-insulin dependent diabetes mellitus (Type II diabetes,NIDDM) usually consists of a combination of diet, exercise, oralhypoglycemic agents, e.g., thiazolidenediones, and, in more severecases, insulin. However, the clinically available hypoglycemic agentscan either have side effects limiting their use, or an agent may not beeffective with a particular patient. In the case of insulin dependentdiabetes mellitus (Type I), insulin administration usually constitutesthe primary course of therapy. Hypoglycemic agents that have fewer sideeffects or succeed where others fail are needed.

[0005] Atherosclerosis, a disease of the arteries, is recognized to bethe leading cause of death in the United States and Western Europe. Thepathological sequence leading to atherosclerosis and occlusive heartdisease is well known. The earliest stage in this sequence is theformation of “fatty streaks” in the carotid, coronary and cerebralarteries and in the aorta. These lesions are yellow in color due to thepresence of lipid deposits found principally within smooth-muscle cellsand in macrophages of the intima layer of the arteries and aorta.Further, it is postulated that most of the cholesterol found within thefatty streaks, in turn, give rise to development of the “fibrousplaque,” which consists of accumulated intimal smooth muscle cells ladenwith lipid and surrounded by extra-cellular lipid, collagen, elastin andproteoglycans. The cells plus matrix form a fibrous cap that covers adeeper deposit of cell debris and more extra cellular lipid. The lipidis primarily free and esterified cholesterol. The fibrous plaque formsslowly, and is likely in time to become calcified and necrotic,advancing to the so-called “complicated lesion”, which accounts for thearterial occlusion and tendency toward mural thrombosis and arterialmuscle spasm that characterize advanced atherosclerosis.

[0006] Epidemiological evidence has firmly established hyperlipidemia asa primary risk factor in causing cardiovascular disease (CVD) due toatherosclerosis. In recent years, medical professionals have placedrenewed emphasis on lowering plasma cholesterol levels, and low-densitylipoprotein cholesterol in particular, as an essential step inprevention of CVD. The upper limits of “normal” are now known to besignificantly lower than heretofore appreciated. As a result, largesegments of Western populations are now realized to be at particularlyhigh risk. Such independent risk factors include glucose intolerance,left ventricular hypertrophy, hypertension, and being of the male sex.Cardiovascular disease is especially prevalent among diabetic subjects,at least in part because of the existence of multiple independent riskfactors in this population. Successful treatment of hyperlipidemia inthe general population, and in diabetic subjects in particular, istherefore of exceptional medical importance.

[0007] Hypertension (high blood pressure) is a condition that occurs inthe human population as a secondary symptom to various other disorderssuch as renal artery stenosis, pheochromocytoma or endocrine disorders.However, hypertension is also evidenced in many patients in whom thecausative agent or disorder is unknown. While such “essential”hypertension is often associated with disorders such as obesity,diabetes and hypertriglyceridemia, the relationship between thesedisorders has not been fully elucidated. Additionally, many patientspresent with symptoms of high blood pressure in the complete absence ofany other signs of disease or disorder.

[0008] It is known that hypertension can directly lead to heart failure,renal failure and stroke (brain hemorrhaging). These conditions arecapable of causing death in a patient. Hypertension can also contributeto the development of atherosclerosis and coronary disease. Theseconditions gradually weaken a patient and can lead to death.

[0009] The exact etiology of “essential” hypertension is unknown, thougha number of factors are believed to contribute to the onset of thedisease. Among such factors are stress, uncontrolled emotions,unregulated hormone release (the renin, angiotensin, aldosteronesystem), excessive salt and water due to kidney malfunction, wallthickening and hypertrophy of the vasculature resulting in constrictedblood vessels, and genetic disposition.

[0010] The treatment of “essential” hypertension has been undertakenbearing the foregoing factors in mind. Thus, a broad range ofbeta-blockers, vasoconstrictors, angiotensin-converting enzyme (ACE)inhibitors, and the like have been developed and marketed asantihypertensives. The treatment of hypertension utilizing thesecompounds has proven beneficial in the prevention of short-intervaldeaths such as heart failure, renal failure, and brain hemorrhaging.However, the development of atherosclerosis or heart disease due tohypertension over a long period of time remains problematic. Thisimplies that although high blood pressure is being reduced, theunderlying cause of essential hypertension is not responding to thistreatment.

[0011] Hypertension has been associated with elevated blood insulinlevels, a condition known as hyperinsulinemia. Insulin, a peptidehormone whose primary actions are to promote glucose utilization,protein synthesis, and the formation and storage of neutral lipids, alsoacts, inter alia, to promote vascular cell growth and increase renalsodium retention. These latter functions can be accomplished withoutaffecting glucose levels and are known causes of hypertension.Peripheral vasculature growth, for example, can cause constriction ofperipheral capillaries while sodium retention increases blood volume.Thus, the lowering of insulin levels in hyperinsulinemics can preventabnormal vascular growth and renal sodium retention caused by highinsulin levels and thereby alleviate hypertension.

[0012] Cardiac hypertrophy is a significant risk factor in thedevelopment of sudden death, myocardial infarction, and congestive heartfailure. These cardiac events are due, at least in part, to increasedsusceptibility to myocardial injury after ischemia and reperfusion thatcan occur in both out-patient and perioperative settings. There iscurrently an unmet medical need to prevent or minimize adversemyocardial perioperative outcomes, particularly perioperative myocardialinfarction. Both non-cardiac and cardiac surgery are associated withsubstantial risks for myocardial infarction or death. Some 7 millionpatients undergoing non-cardiac surgery are considered to be at risk,with incidences of perioperative death and serious cardiac complicationsas high as 20-25% in some series. In addition, of the 400,000 patientsundergoing coronary by-pass surgery annually, perioperative myocardialinfarction is estimated to occur in 5% and death in 1-2%. There iscurrently no marketed drug therapy in this area that reduces damage tocardiac tissue from perioperative myocardial ischemia or enhancescardiac resistance to ischemic episodes. Such a therapy is anticipatedto be life-saving, reduce hospitalizations, enhance quality of life, andreduce overall health care costs of high-risk patients. The mechanism(s)responsible for the myocardial injury observed after ischemia andreperfusion is not fully understood, however, it has been reported (M.F. Allard, et al., Am. J. Physiol., 267: H66-H74 (1994)) that“pre-ischemic glycogen reduction . . . is associated with improvedpost-ischemic left ventricular functional recovery in hypertrophied rathearts.”

[0013] In addition to myocardial ischemia, other tissues can undergoischemia and be damaged resulting in serious problems for the patient.Examples of such tissues include cardiac, brain, liver, kidney, lung,gut, skeletal muscle, spleen, pancreas, nerve, spinal cord, retinatissue, the vasculature, or intestinal tissue.

[0014] Hepatic glucose production is an important target for NIDDMtherapy. The liver is the major regulator of plasma glucose levels inthe post absorptive (fasted) state, and the rate of hepatic glucoseproduction in NIDDM patients is significantly elevated relative tonormal individuals. Likewise, in the postprandial (fed) state, where theliver plays a proportionately smaller role in the total plasma glucosesupply, hepatic glucose production is abnormally high in NIDDM patients.

[0015] Glycogenolysis is an important target for interruption of hepaticglucose production. The liver produces glucose by glycogenolysis(breakdown of the glucose polymer glycogen) and gluconeogenesis(synthesis of glucose from 2- and 3-carbon precursors). Several lines ofevidence indicate that glycogenolysis may make an important contributionto hepatic glucose output in NIDDM. First, in normal post absorptiveman, up to 75% of hepatic glucose production is estimated to result fromglycogenolysis. Second, patients having liver glycogen storage diseases,including Hers' disease (glycogen phosphorylase deficiency), displayepisodic hypoglycemia. These observations suggest that glycogenolysismay be a significant process for hepatic glucose production.

[0016] Glycogenolysis is catalyzed in liver, muscle, and brain bytissue-specific isoforms of the enzyme glycogen phosphorylase. Thisenzyme cleaves the glycogen macromolecule to release glucose-1-phosphateand a new shortened glycogen macromolecule. Several types of glycogenphosphorylase inhibitors have been reported to date: glucose and glucoseanalogs [J. L. Martin, et al., Biochemistry, 30:10101 (1991)]; caffeineand other purine analogs [P. J. Kasvinsky, et al., J. Biol. Chem.,253:3343-3351 and 9102-9106 (1978)]; substitutedN-(indole-2-carbonyl)-amides [PCT Publication Number WO 96/39385]; andsubstituted N-(indole-2-carbonyl)-glycinamides [PCT Publication NumberWO 96/39384]. These compounds, and glycogen phosphorylase inhibitors ingeneral, have been postulated to be of use for the treatment of NIDDM bydecreasing hepatic glucose production and lowering glycemia. [T. B.Blundell, et al., Diabetologia, 35: Suppl. 2, 569-576 (1992) and Martinet al., Biochemistry, 30: 10101 (1991)].

[0017] Myocardial ischemic injury can occur in outpatient as well as inperioperative settings and can lead to the development of sudden death,myocardial infarction, or congestive heart failure. There is currentlyan unmet medical need to prevent or minimize myocardial ischemic injury,particularly perioperative myocardial infarction. Such a therapy isanticipated to be life-saving, reduce hospitalizations, enhance qualityof life, and reduce overall health care costs of high-risk patients.Although there are a variety of hyperglycemia, hypercholesterolemia,hypertension, hyperlipidemia, atherosclerosis and tissue ischemiatherapies, there is a continuing need in the art for alternativetherapies.

SUMMARY OF THE INVENTION

[0018] The present invention provides compounds of formula (I)

[0019] the prodrugs thereof, and the pharmaceutically acceptable saltsof the compounds and prodrugs, wherein R′, R″, R′″, and Z are as definedherein; pharmaceutical compositions thereof; and uses thereof intreating diabetes, insulin resistance, diabetic neuropathy, diabeticnephropathy, diabetic retinopathy, cataracts, hyperglycemia,hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia,atherosclerosis, and tissue ischemia.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides compounds of formula (I)

[0021] the prodrugs thereof, and the pharmaceutically acceptable saltsof the compounds and prodrugs, wherein:

[0022] R′ is

[0023] wherein R represents from 1-3 substituents independently selectedfrom the group consisting of hydrogen; amino; cyano; nitro; halogen;—(C₁-C₆)alkyl; and —(C₁-C₆) alkoxy, wherein the —(C₁-C₆)alkyl group andthe —(C₁-C₆)alkoxy group are each optionally substituted with from 1-6fluorine atoms;

[0024] R″ is

[0025] wherein n represents an integer from 1-3; or

[0026] (ii) —CHR^(a)SO₂(C₁-C₆)alkyl, wherein R^(a) is hydrogen or—(₁-C₆)alkyl;

[0027] R′″ is hydrogen or —(C₁-C₆)alkyl; and

[0028] Z is oxygen or sulfur.

[0029] A generally preferred subgroup of the compounds of formula (I)comprises those compounds wherein:

[0030] R′ is

[0031] wherein R is selected from the group consisting of chloro,fluoro, and methyl;

[0032] R″ is

[0033] R′″ is hydrogen or methyl; and

[0034] Z is oxygen.

[0035] Another generally preferred subgroup of the compounds of formula(I) comprises those compounds wherein:

[0036] R′ is

[0037] wherein R is selected from the group consisting of chloro,fluoro, and methyl;

[0038] R″ is

[0039] R′″ is hydrogen or methyl; and

[0040] Z is oxygen.

[0041] Another generally preferred subgroup of the compounds of formula(I) comprises those compounds wherein:

[0042] R′ is

[0043] wherein R is selected from the group consisting of chloro,fluoro, and methyl;

[0044] R″ is

[0045] R′″ is hydrogen; and

[0046] Z is oxygen.

[0047] Yet another generally preferred subgroup of the compounds offormula (I) comprises those compounds wherein:

[0048] R′ is

[0049] R″ is

[0050] R′″ is methyl; and

[0051] Z is oxygen.

[0052] Yet another generally preferred subgroup of the compounds offormula (I) comprises those compounds wherein:

[0053] R′ is

[0054] R″ is

[0055] R′″ is hydrogen or methyl; and

[0056] Z is oxygen.

[0057] An especially preferred subgroup of the compounds of formula (I)comprises those compounds selected from the group consisting of:

[0058] 5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-hydroxy-3-oxo-propyl]-amide;

[0059] 5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-3-(1,1-dioxo-1-tetrahydro-1-thiophen-2-yl)-2-methoxy-3-oxo-propyl]-amide;

[0060] 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-methoxy-3-oxo-propyl]-amide;

[0061] 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-hydroxy-3-oxo-propyl]-amide;

[0062] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-hydroxy-3-oxo-propyl]-amide;

[0063] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-methoxy-3-oxo-propyl]-amide;and

[0064] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-methoxy-3-oxo-propyl]-amide;the prodrugs thereof, and the pharmaceutically acceptable salts of thecompounds and prodrugs.

[0065] The compounds and intermediates of the present invention may benamed according to either the IUPAC (International Union for Pure andApplied Chemistry) or CAS (Chemical Abstracts Service, Columbus, Ohio)nomenclature systems.

[0066] The carbon atom content of the various hydrocarbon-containingmoieties herein may be indicated by a prefix designating the minimum andmaximum number of carbon atoms in the moiety, for example, the prefix(C_(a)-C_(b))alkyl indicates an alkyl moiety of the integer “a” to “b”carbon atoms, inclusive. Thus, for example, (C₁-C₆) alkyl refers to analkyl group of one to six carbon atoms inclusive, for example, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, andthe like, including all regioisomeric forms thereof, and straight andbranched chain forms thereof.

[0067] The term “alkoxy” refers refers to straight or branched,monovalent, saturated aliphatic chains of carbon atoms bonded to anoxygen atom. Examples of alkoxy groups include methoxy, ethoxy, propoxy,butoxy, iso-butoxy, tert-butoxy, pentoxy, and the like.

[0068] The term “alkyl” refers to straight or branched, monovalent,saturated aliphatic chains of carbon atoms and includes, for example,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl,hexyl, and the like.

[0069] The term “halogen” represents chloro, fluoro, bromo, and iodo.

[0070] The term “mammal” means animals including, for example, dogs,cats, cows, sheep, horses, and humans. Preferred mammals include humans.

[0071] The phrase “pharmaceutically acceptable” indicates that thedesignated carrier, vehicle, diluent, excipient(s), and/or salt must bechemically and/or physically compatible with the other ingredientscomprising the formulation, and physiologically compatible with therecipient thereof.

[0072] The term “prodrug” refers to a compound that is a drug precursorwhich, following administration, releases the drug in vivo via achemical or physiological process (e.g., upon being brought tophysiological pH or through enzyme activity). A discussion of thesynthesis and use of prodrugs is provided by T. Higuchi and W. Stella,“Prodrugs as Novel Delivery Systems, Vol. 14 of the ACS SymposiumSeries, and in Bioreverible Carriers in Drug Design, ed. Edward B.Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0073] The term “radical” denotes a group of atoms that behaves as asingle atom in a chemical reaction, e. g., an organic radical is a groupof atoms that imparts characteristic properties to a compound containingit, or which remains unchanged during a series of reactions, ortransformations.

[0074] The term “salts” refers to organic and inorganic salts of acompound of formula (I), or a stereoisomer, or prodrug thereof. Thesesalts can be prepared in situ during the final isolation andpurification of a compound, or by separately reacting a compound offormula (I), or a stereoisomer or prodrug thereof, with a suitableorganic or inorganic acid or base and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, besylate, palmitate, stearate,laurate, borate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts, as the like.These may also include cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, and thelike, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. For additional examples see,for example, Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0075] The term “substituted” means that a hydrogen atom on a moleculehas been replaced with a different atom or molecule. The atom ormolecule replacing the hydrogen atom is denoted as a “substituent.”

[0076] The symbol “—” represents a covalent bond.

[0077] The phrase “reaction-inert solvent” or “inert solvent” refers toa solvent, or mixture of solvents, that does not interact with startingmaterials, reagents, intermediates, or products in a manner thatadversely affects their desired properties. The terms “treating”,“treated”, or “treatment” as employed herein includes preventative(e.g., prophylactic), palliative, or curative use or result.

[0078] The terms “treating”, “treated”, or “treatment” as employedherein includes preventative (e.g., prophylactic), palliative, orcurative use or result.

[0079] The compounds of formula (I) may contain asymmetric or chiralcenters and, therefore, exist in different stereoisomeric forms. It isintended that all stereoisomeric forms of the compounds of formula (I)as well as mixtures thereof, including racemic mixtures, form part ofthe present invention. In addition, the present invention embraces allgeometric and positional isomers. For example, if a compound of formula(I) incorporates a double bond, both the cis- and trans-forms, as wellas mixtures thereof, are embraced within the scope of the invention.

[0080] Diasteriomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well-known to those of ordinary skill in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diasteriomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diasteriomers and converting (e.g.,hydrolyzing) the individual diasteriomers to the corresponding pureenantiomers. Also, some of the compounds of formula (I) may beatropisomers (e.g., substituted biaryls) and are also considered as partof the invention.

[0081] The compounds of formula (I) may exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents, such as water,ethanol, and the like, and it is intended that the invention embraceboth solvated and unsolvated forms.

[0082] It is also possible that the compounds of formula (I) may existas tautomeric isomers in equilibrium, and all such forms are embracedwithin the scope of the invention.

[0083] The present invention also embraces isotopically-labeledcompounds of formula (I), which are identical to those recited herein,but for the fact that one or more atoms are replaced by an atom havingan atomic mass or mass number different from the atomic mass or massnumber usually found in nature. Examples of isotopes that can beincorporated into compounds of formula (I) include isotopes of hydrogen,carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl,respectively. The compounds of formula (I), the stereoisomers andprodrugs thereof, and the pharmaceutically acceptable salts of thecompounds, stereoisomers, or prodrugs, that contain the aforementionedisotopes and/or other isotopes of the other atoms are intended to bewithin the scope of the instant invention.

[0084] Certain isotopically-labeled compounds of formula (I), forexample those compounds into which radioactive isotopes such as ³H and¹⁴C are incorporated, are useful in compound and/or substrate tissuedistribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their relative ease ofpreparation and facile detection. Furthermore, substitution with heavierisotopes such as deuterium, i.e., ²H, may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life, or reduced dosage requirements and, hence,may be preferred in some circumstances. The isotopically-labeledcompounds of formula (I) can generally be prepared by carrying outprocedures analogous to those disclosed in the Schemes and/or Examplesset forth hereinbelow, by substituting an isotopically-labeled reagentfor a non-isotopically-labeled reagent.

[0085] In another aspect, the invention provides methods of treatingconditions selected from the group consisting of atherosclerosis,diabetes, insulin resistance, diabetic neuropathy, diabetic nephropathy,diabetic retinopathy, cataracts, hypercholesterolemia,hypertriglyceridemia, hyperlipidemia, hyperglycemia, hypertension, andtissue ischemia, including mycardial ischemia, which compriseadministering to a mammal in need of such treatment, a therapeuticallyeffective amount of a compound of formula (I), a prodrug thereof, or apharmaceutically acceptable salt of the compound or prodrug; or apharmaceutical composition comprising a compound of formula (I), or aprodrug thereof, or a pharmaceutically acceptable salt of the compoundor prodrug, and a pharmaceutically acceptable carrier, vehicle, ordiluent. A preferred condition comprises diabetes.

[0086] In another aspect, the invention provides methods for inhibitingglycogen phosphorylase which comprises administering to a mammal in needof such inhibition, a glycogen phosphorylase inhibiting amount of acompound of formula (I), a prodrug thereof, or a pharmaceuticallyacceptable salt of the compound or prodrug; or a pharmaceuticalcomposition comprising a compound of formula (I), or a prodrug thereof,or a pharmaceutically acceptable salt of the compound or prodrug, and apharmaceutically acceptable carrier, vehicle, or diluent.

[0087] The compounds of formula (I) may be administered to a mammal atdosage levels in the range of from about 0.1 mg to about 3,000 mg perday. For a normal adult human having a body mass of about 70 kg, adosage in the range of from about 0.01 mg to about 100 mg per kg bodymass is typically sufficient. However, some variability in the generaldosage range may be required depending upon the age and mass of thesubject being treated, the intended route of administration, theparticular compound being administered, and the like. The determinationof dosage ranges and optimal dosages for a particular mammalian subjectis within the ability of one of ordinary skill in the art having benefitof the instant disclosure.

[0088] According to the methods of the present invention, a compound offormula (I), a prodrug thereof, or a pharmaceutically acceptable salt ofthe compound or prodrug, may be administered in the form of apharmaceutical composition comprising a pharmaceutically acceptablecarrier, vehicle, or diluent. Accordingly, a compound of formula (I), aprodrug thereof, or a pharmaceutically acceptable salt of the compoundor prodrug, may be administered to a subject separately or together inany conventional oral, rectal, transdermal, parenteral (e.g.,intravenous, intramuscular, or subcutaneous), intracisternal,intravaginal, intraperitoneal, intravesical, local (e.g., powder,ointment, or drop), or buccal, or nasal dosage form.

[0089] Pharmaceutical compositions suitable for parenteral injection maycomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions, or emulsions, and sterile powdersfor extemporaneous reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and nonaqueous carriers,vehicles, and diluents include water, ethanol, polyols (such aspropylene glycol, polyethylene glycol, glycerol, and the like), suitablemixtures thereof, vegetable oils (such as olive oil), and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

[0090] The pharmaceutical compositions of the invention may furthercomprise adjuvants, such as preserving, wetting, emulsifying, anddispersing agents. Prevention of microorganism contamination of theinstant compositions can be accomplished with various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,for example, sugars, sodium chloride, and the like. Prolonged absorptionof of injectable pharmaceutical compositions may be effected by the useof agents capable of delaying absorption, for example, aluminummonostearate and gelatin.

[0091] Solid dosage forms for oral administration include capsules,tablets, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert conventional pharmaceuticalexcipient (or carrier) such as sodium citrate or dicalcium phosphate, or(a) fillers or extenders, as for example, starches, lactose, sucrose,mannitol, and silicic acid; (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; (c) humectants, as for example, glycerol; (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid certain complex silicates, andsodium carbonate; (e) solution retarders, as for example, paraffin; (f)absorption accelerators, as for example, quaternary ammonium compounds;(g) wetting agents, as for example, cetyl alcohol and glycerolmonostearate; (h) adsorbents, as for example, kaolin and bentonite;and/or (i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules and tablets, the dosage forms mayfurther comprise buffering agents.

[0092] Solid compositions of a similar type may also be employed asfillers in soft or hard filled gelatin capsules using such excipients aslactose or milk sugar, as well as high molecular weight polyethyleneglycols, and the like.

[0093] Solid dosage forms such as tablets, dragees, capsules, andgranules can be prepared with coatings and shells, such as entericcoatings and others well-known to one of ordinary skill in the art. Theymay also comprise opacifying agents, and can also be of such compositionthat they release the active compound(s) in a delayed, sustained, orcontrolled manner. Examples of embedding compositions that can beemployed are polymeric substances and waxes. The active compound(s) canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

[0094] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosage formmay contain inert diluents commonly used in the art, such as water orother solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, and sesame seed oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, or mixtures of these substances, and the like.

[0095] Besides such inert diluents, the pharmaceutical composition canalso include adjuvants, such as wetting agents, emulsifying andsuspending agents, sweetening, flavoring, and perfuming agents.

[0096] Suspensions, in addition to the active compound(s), may furthercomprise suspending agents, as for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,or mixtures of these substances, and the like.

[0097] Compositions for rectal or vaginal administration preferablycomprise suppositories, which can be prepared by mixing an activecompound(s) with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax, which are solidat ordinary room temperature, but liquid at body temperature, andtherefore, melt in the rectum or vaginal cavity thereby releasing theactive component.

[0098] Dosage forms for topical administration may comprise ointments,powders, sprays and inhalants. The active agent(s) are admixed understerile condition with a pharmaceutically acceptable carrier, vehicle,or diluent, and any preservatives, buffers, or propellants that may berequired.

[0099] The compounds of formula (I) may be prepared according to theexemplary synthetic route disclosed in Scheme I hereinbelow, as well asby other conventional organic preparative methods. It is to beunderstood that the method disclosed in Scheme 1 is intended forpurposes of exemplifying the instant invention, and is not to beconstrued in any manner as a limitation thereon.

[0100] In Scheme 1 hereinabove, an appropriately-substituted1H-indole-2-carboxylic acid derivative (Ia) or6-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid derivative (Ib) iscoupled with: (1) trimethylene sulfone (prepared as described inSynthesis, 7, 582-583 (1982)), tetrahydrothiophene-1,1-dioxide(sulfolane), or tetrahydro-1,1-dioxide-2H-thiopyran (prepared asdescribed in J. Am. Chem. Soc., 114, 3021 (1992)), or (2) an acylicsulfone auxiliary of structural formula H₂CR^(a)SO₂(C₁-C₆)alkyl, whereinR^(a) is hydrogen or —(C₁-C₆)alkyl to afford (I). The coupling istypically effected in an aprotic, non-polar solvent, such as ether ortetrahydrofuran, in the presence of a strong organic base, such aslithium diisopropylamide. The coupling is normally effected belowambient temperature, preferably at, or about, −78° C.

[0101] The substituted indole derivatives of general structure (Ia) maybe prepared according to the methods disclosed in commonly-assigned U.S.Pat. No. 6,297,269, the disclosure of which is incorporated herein byreference. The compounds of general structure (Ib) may be prepared asdescribed hereinbelow in exemplary Preparation 1 or, alternatively,according to the methodologies disclosed in commonly-assigned U.S. Pat.No. 6,399,601, the disclosure of which is incorporated herein byreference.

Preparative Experimental

[0102] Unless otherwise noted, all reactants and reagents were obtainedcommercially. Unless indicated otherwise, the following experimentalabbreviations have the indicated meanings:

[0103] DMF—N,N-dimethylformamide

[0104] HOBT—hydroxybenzotriazole hydrate

[0105] HPLC—high performance liquid chromatography

[0106] hr(s)—hour(s)

[0107] LC/MS—liquid chromatography/mass spectrometry

[0108] min(s)—minute(s)

[0109] THF—tetrahydrofuran

[0110] Preparation 1

[0111] 2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicAcid-[1-benzyl-2-methoxy-2-(methoxymethyl-carbamoyl)-ethyl]-amide (1b,Z=O, R′″=CH₃)

[0112] Triethylamine was added dropwise to a solution of3-amino-2,N-dimethoxy-N-methyl-4-phenyl-butyramide (PCT InternationalApplication Publication No. WO 96/39385) (1.65 g, 5.71 mmol) in DMF (15mL) and cooled in an ice bath. A solution of2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid (U.S. Pat. No.6,399,601) (1.1 g, 5.71 mmol) in 15 mL of DMF was added dropwisefollowed by the addition of HOBT (1.16 g, 11.4 mmol) neat. The reactionmixture was stirred for 5 min andN-ethyliminomethylene-N,N′-dimethyl-propane-1,3-diamine (1.10 g, 5.71mmol) was added neat. The resulting mixture was stirred at roomtemperature for 18 hrs. The reaction mixture was diluted with ethylacetate (150 mL) and washed with saturated ammonium chloride, water, andbrine. The ethyl acetate was dried and evaporated in vacuo to yield 2.54g of crude product. Chromatography over silica gel, eluting with ethylacetate/hexanes (1:2), afforded 1.43 g of the title compound.

EXAMPLE 1 5-Chloro-1H-indole-2-carboxylicAcid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-hydroxy-3-oxo-propyl]-amide

[0113] n-Butyl lithium (2 mL, 2.5 M in hexanes) was added dropwise to asolution of diisopropylamine in THF (5 mL) at 0° C. The reaction mixturewas cooled to −78° C., a THF solution oftetrahydro-1,1-dioxide-2H-thiopyran (0.67 g, 5 mmol) was added dropwise,and then the mixture was warmed to 0° C. To the resulting slurry wasadded 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-2-methoxy-2-(hydroxymethyl-carbamoyl)-ethyl]-amide (PCTInternational Application Publication No. WO 96/39385) (0.42 g, 1 mmol)in 5 mL of THF and the reaction mixture was stirred for 30 min. Thereaction mixture was quenched by the addition of a saturated aqueousammonium chloride solution and extracted with ethyl acetate. Theextracts were dried over magnesium sulfate, filtered, and the solventremoved in vacuo to afford 33 mg of the title compound. LC/MS (E+1) 490

[0114] The following compounds of formula (I) were prepared in a manneranalogous to that described in Example 1:

[0115] 5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-hydroxy-3-oxo-propyl]-amide—LC/MS(E+1) 476;

[0116] 5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-3-(1,1-dioxo-1-tetrahydro-1-thiophen-2-yl)-2-methoxy-3-oxo-propyl]-amide—LC/MS(E+1) 490;

[0117] 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-1-thietan-2-yl)-2-hydroxy-3-oxo-propyl]-amide—LC/MS(E+1) 462;

[0118] 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-methoxy-3-oxo-propyl]-amide—LC/MS(E+1) 504;

[0119] 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-1-thietan-2-yl)-2-methoxy-3-oxo-propyl]-amide—LC/MS(E+1) 476;

[0120] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-hydroxy-3-oxo-propyl]-amide—LC/MS(E+1) 482;

[0121] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-methoxy-3-oxo-propyl]-amide—LC/MS(E+1) 496;

[0122] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-methoxy-3-oxo-propyl]-amide—LC/MS(E+1) 510; and

[0123] 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-1-thietan-2-yl)-2-methoxy-3-oxo-propyl]-amide—LC/MS(E+1) 482.

EXAMPLE 2 5-Chloro-1H-indole-2-carboxylic Acid(1-benzyl-4-ethanesulfonyl-2-hydroxy-3-oxo-butyl)-amide

[0124] A solution of methanesulfonyl-ethane (1.08 gms; 10 mmol) in 5 mLof THF was added dropwise to a 1 M lithium diisopropylamide (LDA)solution in 10 mL of THF at −78° C. The solution was then warmed to 0°C. To the resulting slurry was added a solution of5-chloro-1H-indole-2-carboxylic acid{1-[hydroxy-(methoxy-methyl-carbamoyl)-methyl]-2-phenyl-ethyl}-amide(0.42 gms; 1 mmol) in 5 mL of THF and the reaction mixture stirred for30 min. The reaction mixture was poured into an ice-cold mixture ofethyl acetate (50 mL)/1 N HCl (50 mL). The organic layer was separated,dried, and the solvent removed in vacuo to yield 552 mg of a foam.Further purification over a C-8 HPLC column (water, formic acid,acetonitrile) afforded 255 mg of the title product. (LC/MS ES+463)

[0125] The following compounds of formula (I) were prepared in a manneranalogous to that described in Example 2:

[0126] 5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-2-hydroxy-4-methanesulfonyl-3-oxo-butyl)-amide—LC/MS(E+1) 449; and

[0127] 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-2-hydroxy-3-oxo-4-(propane-2-sulfonyl)-butyl]-amide LC/MS(E+1) 477.

Biological Protocols

[0128] The utility of the compounds of formula (I), the prodrugsthereof, and the pharmaceutically acceptable salts of the compounds andprodrugs, in the treatment or prevention of diseases (such as aredetailed herein) in animals, particularly mammals (e.g., humans) may bedemonstrated by the activity thereof in conventional assays known to oneof ordinary skill in the relevant art, including the in vitro and invivo assays described below. Such assays also provide a means wherebythe activities of the compounds of formula (I) can be compared with theactivities of other known compounds.

[0129] Glycogen Phosphorylase Production and Assays

[0130] The three different purified glycogen phosphorylase (GP)isoenzymes, wherein glycogen phosphorylase is in the activated “a” state(referred to as glycogen phosphorylase a, or the abbreviation GPa), andreferred to here as human liver glycogen phosphorylase a (HLGPa), humanmuscle glycogen phosphorylase a (HMGPa), and human brain glycogenphosphorylase a (HBGPa), can be obtained according to the followingprocedures.

[0131] Expression and Fermentation

[0132] The HLGP cDNAs (obtained as described in Newgard, et al., Proc.Natl. Acad. Sci., 83, 8132-8136 (1986), and Newgard, et al., Proc. Natl.Acad. Sci., 263, 3850-3857 (1988), respectively) and HMGP cDNAs(obtained by screening a Stratagene (Stratagene Cloning Systems, LaJolla, Calif.) human muscle cDNA library with a polymerase chainreaction (PCR)-generated cDNA fragment based on information andmethodology reported for isolation of the human skeletal muscle glycogenphosphorylase gene and partial cDNA sequence by Kubisch, et al., Centerfor Molecular Neurobiology, University of Hamburg, Martinistrasse 85,Hamburg, 20246 Germany; Genbank (National Center for BiotechnologyInformation, National Institutes of Health, USA) Accession NumbersU94774, U94775, U94776 and U94777, submitted Mar. 20, 1997; Burke, etal., Proteins, 2, 177-187 (1987); and Hwang et al., Eur. J. Biochem.,152, 267-274 (1985)) are expressed from plasmid pKK233-2 (PharmaciaBiotech. Inc., Piscataway, N.J.) in E. coli strain XL-1 Blue (StratageneCloning Systems, LaJolla, Calif.). The strain is inoculated into LBmedium (consisting of 10 g tryptone, 5 g yeast extract, 5 g NaCl, and 1ml 1N NaOH per liter) plus 100 mg/L ampicillin, 100 mg/l pyridoxine and600 mg/L MnCl₂ and grown at 37° C. to a cell density of OD₅₅₀=1.0. Atthis point, the cells are induced with 1 mMisopropyl-1-thio-β-D-galactoside (IPTG). Three hours after induction thecells are harvested by centrifugation and cell pellets are frozen at−70° C. until needed for purification.

[0133] The HBGP cDNA can be expressed by several methodologies, forexample, by the method described by Crerar, et al., J. Biol. Chem. 270,13748-13756 (1995), wherein the method for the expression of HBGP is asfollows: the HBGP cDNA can be expressed from plasmid pTACTAC in E. colistrain 25A6. The strain is inoculated into LB medium (consisting of 10 gtryptone, 5 g yeast extract, 5 g NaCl, and 1 ml 1N NaOH per liter) plus50 mg/L ampicillin and grown overnight, then resuspended in fresh LBmedium plus 50 mg/L ampicillin, and reinoculated into a 40× volume ofLB/ampicillin media containing 250 μM isopropyl-1-thio-β-D-galactoside(IPTG), 0.5 mM pyridoxine and 3 mM MnCl₂ and grown at 22° C. for 48-50hours. The cells can then be harvested by centrifugation and cellpellets are frozen at −70° C. until needed for purification.

[0134] Alternatively, the HLGP and HBGP cDNAs are expressed from plasmidpBlueBac III (Invitrogen Corp., San Diego, Calif.) which iscotransfected with BaculoGold Linear Viral DNA (Pharmingen, San Diego,Calif.) into Sf9 cells. Recombinant virus is subsequentlyplaque-purified. For production of protein, Sf9 cells grown inserum-free medium (Sf-900 II serum free medium, Gibco BRL, LifeTechnologies, Grand Island, N.Y.) are infected at an moi of 0.5 and at acell density of 2×10⁶ cells/ml. After growth for 72 hours at 27° C.,cells are centrifuged, and the cell pellets frozen at −70° C. untilneeded for purification.

[0135] Purification of Glycogen Phoshorylase Expressed in E. coli

[0136] The E. coli cells in pellets described above are resuspended in25 mM β-glycerophosphate (pH 7.0) with 0.2 mM DTT, 1 mM MgCl₂, plus thefollowing protease inhibitors: 0.7 μg/ml Pepstatin A 0.5 μg/ml Leupeptin0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM EDTA,

[0137] lysed by pretreatment with 200 μg/ml lysozyme and 3 μg/ml DNAasefollowed by sonication in 250 ml batches for 5×1.5 minutes on ice usinga Branson Model 450 ultrasonic cell disrupter (Branson Sonic Power Co.,Danbury Conn.). The E. coli cell lysates are then cleared bycentrifugation at 35,000× g for one hour followed by filtration through0.45 micron filters. GP in the soluble fraction of the lysates(estimated to be less than 1% of the total protein) is purified bymonitoring the enzyme activity (as described in GPa Activity Assaysection, below) from a series of chromatographic steps detailed below.

[0138] Immobilized Metal Affinity Chromatography (IMAC)

[0139] This step is based on the method of Luong, et al., Journal ofChromatography, 584, 77-84 (1992). Five hundred ml of the filteredsoluble fraction of cell lysates (prepared from approximately 160-250 gof original cell pellet) are loaded onto a 130 ml column of IMACChelating-Sepharose (Pharmacia LKB Biotechnology, Piscataway, N.J.)which has been charged with 50 mM CuCl₂ and 25 mM β-glycerophosphate,250 mM NaCl and 1 mM imidazole at pH 7 (equilibration buffer). Thecolumn is washed with equilibration buffer until the A₂₈₀ returns tobaseline. The sample is then eluted from the column with the same buffercontaining 100 mM imidazole to remove the bound GP and other boundproteins. Fractions containing the GP activity are pooled (approximately600 ml), and ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol(DTT), phenylmethylsulfonyl fluoride (PMSF), leupeptin and pepstatin Aare added to obtain 0.3 mM, 0.2 mM, 0.2 mM, 0.5 μg/ml and 0.7 μg/mlconcentrations respectively. The pooled GP is desalted over a SephadexG-25 column (Sigma Chemical Co., St. Louis, Mo.) equilibrated with 25 mMTris-HCl (pH 7.3), 3 mM DTT buffer (Buffer A) to remove imidazole and isstored on ice and subjected to a second chromatographic step (below) ifnecessary.

[0140] 5′-AMP-Sepharose Chromatography

[0141] The desalted pooled GP sample (approximately 600 mL) is thenmixed with 70 ml of 5′-AMP Sepharose (Pharmacia LKB Biotechnology,Piscataway, N.J.) which has been equilibrated with Buffer A (see above).The mixture is gently agitated for one hour at 22° C. then packed into acolumn and washed with Buffer A until the A₂₈₀ returns to baseline. GPand other proteins are eluted from the column with 25 mM Tris-HCl, 0.2mM DTT and 10 mM adenosine 5′-monophosphate (AMP) at pH 7.3 (Buffer B).GP-containing fractions are pooled following identification bydetermining enzyme activity described below and visualizing the M_(r)approximately 97 kdal GP protein band by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by silverstaining (2D-silver Stain II “Daiichi Kit”, Daiichi Pure Chemicals Co.,LTD., Tokyo, Japan) and then pooled. The pooled GP is dialyzed into 25mM β-glycerophosphate, 0.2 mM DTT, 0.3 mM EDTA, 200 mM NaCl, pH 7.0buffer (Buffer C) and stored on ice until use.

[0142] Prior to use of the GP enzyme, the enzyme is converted from theinactive form as expressed in E. coli strain XL-1 Blue (designated GPb)(Stragene Cloning Systems, La Jolla, Calif.), to the active form(designated GPa) by the procedure described in Section (A) Activation ofGP below.

[0143] Purification of Glycogen Phosphorylase Expressed in Sf9 Cells

[0144] The Sf9 cells in pellets described above are resuspended in 25 mMβ-glycerophosphate (pH 7.0) with 0.2 mM DTT, 1 mM MgCl2, plus thefollowing protease inhibitors: 0.7 μg/ml Pepstatin A 0.5 μg/ml Leupeptin0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM EDTA,

[0145] lysed by pretreatment with 3 μg/ml DNAase followed by sonicationin batches for 3×1 minutes on ice using a Branson Model 450 ultrasoniccell disrupter (Branson Sonic Power Co., Danbury Conn.). The Sf9 celllysates are then cleared by centrifugation at 35,000×g for one hourfollowed by filtration through 0.45 micron filters. GP in the solublefraction of the lysates (estimated to be 1.5% of the total protein) ispurified by monitoring the enzyme activity (as described in GPa ActivityAssay section, below) from a series of chromatographic steps detailedbelow.

[0146] Immobilized Metal Affinity Chromatography (IMAC)

[0147] Immobilized Metal Affinity Chromatography is performed asdescribed in the section above. The pooled, desalted GP is then storedon ice until further processed.

[0148] Activation of GP

[0149] Before further chromatography, the fraction of inactive enzyme asexpressed in Sf9 cells (designated GPb) is converted to the active form(designated GPa) by the following procedure described in Section (A)Activation of GP below.

[0150] Anion Exchange Chromatography

[0151] Following activation of the IMAC purified GPb to GPa by reactionwith the immobilized phosphorylase kinase, as described below, thepooled GPa fractions are dialyzed against 25 mM Tris-HCl, pH 7.5,containing 0.5 mM DTT, 0.2 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride(PMSF), 1.0 μg/ml leupeptin and 1.0 μg/ml pepstatin A. The fraction isthen loaded onto a MonoQ Anion Exchange Chromatography column (PharmaciaBiotech. Inc., Piscataway, N.J.). The column is washed withequilibration buffer until the A₂₈₀ returns to baseline. The sample isthen eluted from the column with a linear gradient of 0-0.25 M NaCl toremove the bound GP and other bound proteins. GP-containing fractionselute between 0.1-0.2 M NaCl range, as detected by monitoring the eluantfor peak protein absorbance at A₂₈₀. The GP protein is then identifiedby visualizing the M_(r) approximately 97 kdal GP protein band by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followedby silver staining (2D-silver Stain II “Daiichi Kit”, Daiichi PureChemicals Co., LTD., Tokyo, Japan) and then pooled. The pooled GP isdialyzed into 25 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid(BES), 1.0 mM DTT, 0.5 mM EDTA, 5 mM NaCl, pH 6.8 buffer and stored onice until use.

[0152] Determination of GP Enzyme Activity

[0153] A) Activation of GP: Conversion of GPb to GPa

[0154] Prior to the determination of GP enzyme activity, the enzyme isconverted from the inactive form as expressed in E. coli strain XL-1Blue (designated GPb) (Stragene Cloning Systems, La Jolla, Calif.), tothe active form (designated GPa) by phosphorylation of GP usingphosphorylase kinase as follows. The fraction of inactive enzyme asexpressed in Sf9 cells (designated GPb) is also converted to the activeform (designated GPa) by the follow procedure.

[0155] GP Reaction with Immobilized Phosphorylase Kinase

[0156] Phosphorylase kinase (Sigma Chemical Company, St. Louis, Mo.) isimmobilized on Affi-Gel® 10 (BioRad Corp., Melville, N.Y.) in accordancewith the manufacturer's instructions. In brief, the phosphorylase kinaseenzyme (10 mg) is incubated with washed Affi-Gel® beads (1 ml) in 2.5 mlof 100 mM HEPES and 80 mM CaCl₂ at pH 7.4 for 4 hours at 4° C. TheAffi-Gel® beads are then washed once with the same buffer prior toblocking with 50 mM HEPES and 1 M glycine methyl ester at pH 8.0 for onehour at room temperature. Blocking buffer is removed and replaced with50 mM HEPES (pH 7.4), 1 mM β-mercaptoethanol and 0.2% NaN₃ for storage.Prior to use to convert GPb to GPa, the Affi-Gel® immobilizedphosphorylase kinase beads are equilibrated by washing in the bufferused to perform the kinase reaction, consisting of 25 mMβ-glycerophosphate, 0.3 mM DTT, and 0.3 mM EDTA at pH 7.8 (kinase assaybuffer).

[0157] The partially purified, inactive GPb obtained from5′-AMP-Sepharose chromatography above (from E. coli) or the mixture ofGPa and GPb obtained from IMAC above (from Sf9 cells) is diluted 1:10with the kinase assay buffer then mixed with the aforementionedphosphorylase kinase enzyme immobilized on the Affi-Gel® beads. NaATP isadded to 5 mM and MgCl₂ to 6 mM. The resulting mixture is mixed gentlyat 25° C. for 30 to 60 minutes. The activated sample is removed from thebeads and the percent activation of GPb by conversion to GPa isestimated by determining GP enzyme activity in the presence and absenceof 3.3 mM AMP. The percentage of total GP enzyme activity due to GPaenzyme activity (AMP-independent) is then calculated as follows:${\% \quad {of}\quad {total}\quad {HLGPa}} = \frac{{{HLGP}\quad {activity}} - {AMP}}{{{HLGP}\quad {activity}} + {AMP}}$

[0158] Alternately, the conversion of GPb to GPa can be monitored byisoelectric focusing, based on the shift in electrophoretic mobilitynoted following conversion of GPb to GPa. GP samples are analyzed byisoelectric focusing (IEF) utilizing the Pharmacia PfastGel System(Pharmacia Biotech. Inc., Piscataway, N.J.) using precast gels (pI range4-6.5) according to the manufacturer's recommended method. The resolvedGPa and GPb bands are then visualized on the gels by silver staining(2D-silver Stain II “Daiichi Kit”, Daiichi Pure Chemicals Co., LTD.,Tokyo, Japan). Identification of GPa and GPb is made by comparison to E.coli derived GPa and GPb standards run in parallel on the same gels asthe experimental samples.

[0159] B) GPa Activity Assay

[0160] The disease/condition treating/preventing activities describedherein of the compounds of formula (I) can be indirectly determined byassessing the effect of the compounds of formula (I) on the activity ofthe activated form of glycogen phosphorylase (GPa) by one of twomethods: (1) GPa activity is measured in the forward direction bymonitoring the production of glucose-1-phosphate from glycogen, or (2)by following the reverse reaction, measuring glycogen synthesis fromglucose-1-phosphate by the release of inorganic phosphate. All reactionsare run in triplicate in 96-well microtiter plates, and the change inabsorbance due to formation of the reaction product is measured at thewavelength specified below in a MCC/340 MKII Elisa Reader (Lab Systems,Finland), connected to a Titertech Microplate Stacker (ICN BiomedicalCo, Huntsville, Ala.).

[0161] To measure the GPa enzyme activity in the forward direction, theproduction of glucose-1-phosphate from glycogen is monitored by themultienzyme coupled general method of Pesce et al., Clinical Chemistry23, 1711-1717 (1977) modified as follows: 1 to 100 μg GPa, 10 unitsphosphoglucomutase and 15 units glucose-6-phosphate dehydrogenase(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) are diluted to 1mL in Buffer D (pH 7.2, 50 mM HEPES, 100 mM KCl, 2.5 mMethyleneglycoltetraacetic acid (EGTA), 2.5 mM MgCl₂, 3.5 mM KH₂PO₄ and0.5 mM dithiothreitol). Twenty μl of this stock is added to 80 μl ofBuffer D containing 0.47 mg/mL glycogen, 9.4 mM glucose, 0.63 mM of theoxidized form of nicotinamide adenine dinucleotide phosphate (NADP+).The formula (I) compound to be tested is added as 5 μl, of solution in14% dimethylsulfoxide (DMSO) prior to the addition of the enzymes. Thebasal rate of GPa enzyme activity in the absence of inhibitors, e.g., acompound of formula (I), is determined by adding 5 μl, of 14% DMSO and afully-inhibited rate of GPa enzyme activity is obtained by adding 20 μl,of 50 mM of the positive control test substance, caffeine. The reactionis followed at room temperature by measuring the conversion of oxidizedNADP+ to reduced NADPH at 340 nm.

[0162] To measure the GPa enzyme activity in the reverse direction, theconversion of glucose-1-phosphate into glycogen plus inorganic phosphateis measured by the general method described by Engers, et al., Can. J.Biochem., 48, 746-754 (1970) modified as follows: 1 to 100 μg GPa isdiluted to 1 ml in Buffer E (pH 7.2, 50 mM HEPES, 100 mM KCl, 2.5 mMEGTA, 2.5 mM MgCl₂ and 0.5 mM dithiothreitol). Twenty μl of this stockis added to 80 μl of Buffer E with 1.25 mg/ml glycogen, 9.4 mM glucose,and 0.63 mM glucose-1-phosphate. The formula (I) compound to be testedis added as 5 μl of solution in 14% DMSO prior to the addition of theenzyme. The basal rate of GPa enzyme activity in the absence of addedinhibitors, e.g., a compound of formula (I), is determined by adding 5μl of 14% DMSO and a fully-inhibited rate of GPa enzyme activity isobtained by adding 20 μL of 50 mM caffeine. This mixture is incubated atroom temperature for 1 hour and the inorganic phosphate released fromthe glucose-1-phosphate is measured by the general method of Lanzetta etal., Anal. Biochem., 100, 95-97 (1979)] modified as follows: 150 μl of10 mg/ml ammonium molybdate, 0.38 mg/ml malachite green in 1 N HCl isadded to 100 μl of the enzyme mix. After a 20 minute incubation at roomtemperature, the absorbance is measured at 620 nm.

[0163] The above assays, carried out with a range of concentrations offormula (I) compounds, allows the determination of an IC₅₀ value(concentration of a compound required for 50% inhibition) for the invitro inhibition of GPa enzyme activity by that compound.

[0164] The compounds of formula (I) are readily adapted to clinical useas hypoglycemic agents. The hypoglycemic activity of the compounds offormula (I) can be determined by the amount of a formula (I) compoundthat reduces glucose levels relative to a vehicle without a formula (I)compound in male ob/ob mice. The test also allows the determination ofan approximate minimal effective dose (MED) value for the in vivoreduction of plasma glucose concentration in such mice for such formula(I) compounds.

[0165] Since the concentration of glucose in blood is closely related tothe development of diabetic disorders, the compounds of formula (I), byvirtue of their hypoglycemic action, prevent, arrest and/or regressdiabetic disorders.

[0166] Five to eight week old male C57BL/6J-ob/ob mice (JacksonLaboratory, Bar Harbor, Me.) are housed five per cage under standardanimal care practices. After a one-week acclimation period, the animalsare weighed and 25 microliters of blood are collected from theretro-orbital sinus prior to any treatment. The blood sample isimmediately diluted 1:5 with saline containing 0.025% sodium heparin,and held on ice for metabolite analysis. Animals are assigned totreatment groups so that each group has a similar mean for plasmaglucose concentration. After group assignment, animals are dosed orallyeach day for four days with the vehicle consisting of either: (1) 0.25%w/v methyl cellulose in water without pH adjustment; or (2) 0.1%Pluronic® P105 Block Copolymer Surfactant (BASF Corporation, Parsippany,N.J.) in 0.1% saline without pH adjustment. On day 5, the animals areweighed again and then dosed orally with a formula (I) compound, or thevehicle alone. All compounds are administered in vehicle consisting ofeither: (1) 0.25% w/v methyl cellulose in water; (2) 10% DMSO/0.1%Pluronic® in 0.1% saline without pH adjustment; or 3) neat PEG 400without pH adjustment. The animals are then bled from the retro-orbitalsinus three hours later for determination of blood metabolite levels.The freshly collected samples are centrifuged for two minutes at10,000×g at room temperature. The supernatant is analyzed for glucose,for example, by the Abbott VP™ (Abbott Laboratories, DiagnosticsDivision, Irving, Tex.) and VP Super System® Autoanalyzer (AbbottLaboratories, Irving, Tex.), or by the Abbott Spectrum CCX™ (AbbottLaboratories, Irving, Tex.) using the A-Gent™Glucose-UV Test reagentsystem (Abbott Laboratories, Irving, Tex.) (a modification of the methodof Richterich and Dauwalder, Schweizerische Medizinische Wochenschrift,101, 860 (1971)) (hexokinase method) using a 100 mg/dl standard. Plasmaglucose is then calculated using the following equation:

Plasma glucose (mg/dl)=Sample value×8.14

[0167] where 8.14 is the dilution factor, adjusted for plasma hematocrit(assuming the hematocrit is 44%).

[0168] The animals dosed with vehicle maintain substantially unchangedhyperglycemic glucose levels (e.g., greater than or equal to 250 mg/dl),animals treated with compounds having hypoglycemic activity at suitabledoses have significantly depressed glucose levels. Hypoglycemic activityof the compounds of formula (I) is determined by statistical analysis(unpaired t-test) of the mean plasma glucose concentration between thetest compound group and vehicle-treated group on day 5. The above assaycarried out with a range of doses of a formula (I) compound allows thedetermination of an approximate minimal effective dose (MED) value forthe in vivo reduction of plasma glucose concentration.

[0169] The compounds of formula (I) are readily adapted to clinical useas hyperinsulinemia reversing agents, triglyceride lowering agents andhypocholesterolemic agents. Such activity can be determined by theamount of the compound of formula (I) that reduces insulin,triglycerides or cholesterol levels relative to a control vehiclewithout test compound in male ob/ob mice.

[0170] Since the concentration of cholesterol in blood is closelyrelated to the development of cardiovascular, cerebral vascular orperipheral vascular disorders, the compounds of formula (I), by virtueof their hypocholesterolemic action, prevent, arrest and/or regressatherosclerosis.

[0171] Since the concentration of insulin in blood is related to thepromotion of vascular cell growth and increased renal sodium retention,(in addition to the other actions, e.g., promotion of glucoseutilization) and these functions are known causes of hypertension, thecompounds of formula (I), by virtue of their hypoinsulinemic action,prevent, arrest and/or regress hypertension.

[0172] Since the concentration of triglycerides in blood contributes tothe overall levels of blood lipids, the compounds of formula (I), byvirtue of their triglyceride lowering and/or free fatty acid loweringactivity prevent, arrest and/or regress hyperlipidemia.

[0173] Five to eight week old male C57BU6J-ob/ob mice (JacksonLaboratory, Bar Harbor, Me.) are housed five per cage under standardanimal care practices and fed standard rodent diet ad libitum. After aone-week acclimation period, the animals are weighed and 25 microlitersof blood are collected from the retro-orbital sinus prior to anytreatment. The blood sample is immediately diluted 1:5 with salinecontaining 0.025% sodium heparin, and held on ice for plasma glucoseanalysis. Animals are assigned to treatment groups so that each grouphas a similar mean for plasma glucose concentration. The compound offormula (I) to be tested is administered by oral gavage as anapproximately 0.02% to 2.0% solution (w/v) in either: (1) 10% DMSO/0.1%Pluronic® P105 Block Copolymer Surfactant (BASF Corporation, Parsippany,N.J.) in 0.1% saline without pH adjustment, or (2) 0.25% w/vmethylcellulose in water without pH adjustment. Alternatively, thecompound of formula (I) may be dissolved or suspended in neat PEG 400,and administered by oral gavage. Single daily dosing (s.i.d.), twicedaily dosing (b.i.d.), or thrice daily dosing (t.i.d.) is maintained,for example, 1 to 28 days. Control mice receive the 10% DMSO/0.1%Pluronice P105 in 0.1% saline without pH adjustment, or the 0.25% w/vmethylcellulose in water without pH adjustment, or the neat PEG 400without pH adjustment.

[0174] One to three hours after the last dose is administered, theanimals are sacrificed by decapitation and trunk blood is collected in0.5 ml serum separator tubes containing 3.6 mg of a 1:1 weight/weightsodium fluoride:potassium oxalate mixture. The freshly collected samplesare centrifuged for two minutes at 10,000×g at room temperature, and theserum supernatant is transferred and diluted 1:1 volume/volume with a1TIU/ml aprotinin solution in 0.1% saline without pH adjustment.

[0175] The diluted serum samples are then stored at −80° C. untilanalysis. The thawed, diluted serum samples are analyzed for insulin,triglycerides, free fatty acids and cholesterol levels. Serum insulinconcentration is determined using Equate® RIA INSULIN kits (doubleantibody method; as specified by the manufacturer) available from Binax,South Portland, Me. The inter assay coefficient of variation is ≦10%.Serum triglycerides are determined using the Abbott VP™ and VP SuperSystem® Autoanalyzer (Abbott Laboratories, Irving, Tex.), or the AbbottSpectrum CCX™ (Abbott Laboratories, Irving, Tex.) using the A-Gent™Triglycerides Test reagent system (Abbott Laboratories, DiagnosticsDivision, Irving, Tex.) (lipase-coupled enzyme method; a modification ofthe method of Sampson, et al., Clinical Chemistry, 21, 1983 (1975)).Serum or plasma total cholesterol levels are determined using the AbbottVP™ and VP Super System® Autoanalyzer (Abbott Laboratories, Irving,Tex.), and A-Gent™ Cholesterol Test reagent system (cholesterolesterase-coupled enzyme method; a modification of the method of Allain,et al., Clinical Chemistry, 20, 470 (1974)) using 100 and 300 mg/dlstandards. Serum or plasma free fatty acid concentration is determinedutilizing a kit from Amano International Enzyme Co., Inc., as adaptedfor use with the Abbott VP™ and VP Super System® Autoanalyzer (AbbottLaboratories, Irving, Tex.), or the Abbott Spectrum CCX™ (AbbottLaboratories, Irving, Tex.). Serum or plasma insulin, triglycerides,free fatty acids, and total cholesterol levels are then calculated usingthe following equations:

Serum or plasma insulin (μU/ml)=Sample value×2

Serum or plasma triglycerides (mg/dl)=Sample value×2

Serum or plasma total cholesterol (mg/dl)=Sample value×2

Serum or plasma free fatty acid (μEq/l)=Sample value×2

[0176] where 2 is the dilution factor.

[0177] The animals dosed with vehicle maintain substantially unchanged,elevated serum or plasma insulin (e.g., 275 μU/ml), serum or plasmatriglycerides (e.g., 235 mg/dl), serum or plasma free fatty acid (1500mEq/ml) and serum or plasma total cholesterol (e.g., 190 mg/dl) levels,while animals treated with compounds of formula (I) generally displayreduced serum or plasma insulin, triglycerides, free fatty acid, andtotal cholesterol levels. The serum or plasma insulin, triglycerides,free fatty acid, and total cholesterol lowering activity of thecompounds of formula (I) are determined by statistical analysis(unpaired t-test) of the mean serum or plasma insulin, triglycerides, ortotal cholesterol concentration between the formula (I) compound groupand the vehicle-treated control group.

1. A compound of formula (I)

the prodrugs thereof, and the pharmaceutically acceptable salts of saidcompounds and prodrugs, wherein: R′ is

wherein R represents from 1-3 substituents independently selected fromthe group consisting of hydrogen; amino; cyano; nitro; halogen;—(C₁-C₆)alkyl; and —(C₁-C₆) alkoxy, wherein said —(C₁-C₆)alkyl group andsaid —(₁-C₆)alkoxy group are each optionally substituted with from 1-6fluorine atoms; R″ is

wherein n represents an integer from 1-3; or (ii)—CHR^(a)SO₂(C₁-C₆)alkyl, wherein R^(a) is hydrogen or —(C₁-C₆)alkyl; R′″is hydrogen or —(C₁-C₆)alkyl; and Z is oxygen or sulfur.
 2. A compoundof claim 1, wherein: R′ is

wherein R is selected from the group consisting of chloro, fluoro, andmethyl; R″ is

R′″ is hydrogen or methyl; and Z is oxygen.
 3. A compound of claim 1,wherein: R′ is

wherein R is selected from the group consisting of chloro, fluoro, andmethyl; R″ is

R′″ is hydrogen or methyl; and Z is oxygen.
 4. A compound of claim 1,wherein: R′ is

wherein R is selected from the group consisting of chloro, fluoro, andmethyl; R″ is

R′″ is hydrogen; and Z is oxygen.
 5. A compound of claim 1 selected fromthe group consisting of: 5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-hydroxy-3-oxo-propyl]-amide;5-chloro-1H-indole-2-carboxylicacid-(1-benzyl-3-(1,1-dioxo-1-tetrahydro-1-thiophen-2-yl)-2-methoxy-3-oxo-propyl]-amide;5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-methoxy-3-oxo-propyl]-amide;and 5-chloro-1H-indole-2-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-hydroxy-3-oxo-propyl]-amide;the prodrugs thereof, and the pharmaceutically acceptable salts of thecompounds and prodrugs.
 6. A compound of claim 1, wherein R′ is

R″ is

R′″ is methyl; and Z is oxygen.
 7. A compound of claim 1, wherein: R′ is

R″ is

R′″ is hydrogen or methyl; and Z is oxygen.
 8. A compound of claim 1selected from the group consisting of:2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-hydroxy-3-oxo-propyl]-amide;2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-tetrahydro-1-thiophen-2-yl)-2-methoxy-3-oxo-propyl]-amide;and 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylicacid-[1-benzyl-3-(1,1-dioxo-hexahydro-1-thiopyran-2-yl)-2-methoxy-3-oxo-propyl]-amide;the prodrugs thereof, and the pharmaceutically acceptable salts of saidcompounds and prodrugs.
 9. A pharmaceutical composition comprising acompound of claim 1, a prodrug thereof, or a pharmaceutically acceptablesalt of said compound or said prodrug; and a pharmaceutically acceptablecarrier, vehicle, or diluent.
 10. A method of treating a conditionselected from the group consisting of atherosclerosis, diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, cataracts, hypercholesterolemia, hypertriglyceridemia,hyperlipidemia, hyperglycemia, hypertension, tissue ischemia, andmycardial ischemia, which comprises administering to a mammal in need ofsuch treatment, a therapeutically effective amount of a compound ofclaim 1, a prodrug thereof, or a pharmaceutically acceptable salt ofsaid compound or said prodrug; or a pharmaceutical compositioncomprising said compound of claim 1, or said prodrug thereof, or saidpharmaceutically acceptable salt of said compound or said prodrug, and apharmaceutically acceptable carrier, vehicle, or diluent.
 11. A methodof claim 10 wherein said condition is diabetes.
 12. A method ofinhibiting glycogen phosphorylase which comprises administering to amammal in need of such inhibition, a glycogen phosphorylase inhibitingamount of a compound of claim 1, a prodrug thereof, or apharmaceutically acceptable salt of said compound or said prodrug; or apharmaceutical composition comprising said compound of claim 1, or saidprodrug thereof, or said pharmaceutically acceptable salt of saidcompound or said prodrug, and a pharmaceutically acceptable carrier,vehicle, or diluent.